PubMed · 8897308
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C Ercoli, T C Abrahamsen. 1996. A simple method for making accurate duplicate casts.. https://doi.org/10.1016/s0022-3913(96)90556-9
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Transplantation of immunoisolated islets of Langerhans has been proposed as a promising approach to treating insulin-dependent diabetes mellitus. Recently, a cell delivery system based on a multicomponent microcapsule has been designed for the immunoisolation of insulin-secreting pancreatic islets. The capsule, formed by polyelectrolyte complexation of sodium alginate and cellulose sulfate with poly(methylene-co-guanidine), markedly has improved mechanical strength compared with the widely used alginate/poly(L-lysine) capsules. It also provides a flexibility for readily adjusting membrane thickness and capsule size, and, more important, the membrane permeability can be altered over a wide range of molecular sizes. To rigorously test the capsule diffusion properties, we have improved capsule permeability measurement by using two complementary methods: (1) size exclusion chromatography with dextran standards; and (2) newly developed methodology for assessing permeability to a series of biologically relevant proteins. Viability and function of rat pancreatic islets enclosed in the capsules with different permeability were tested in vitro. The insulin secretion of encapsulated islets was well preserved even though slightly delayed in comparison with a control group of free islets. We believe that the unique features of this encapsulation system together with the precise characterization of its physical parameters will enable us to find the optimal range of capsule permeability for in vitro and in vivo survival and function of encapsulated pancreatic islets.
STUDY DESIGN: Cells from normal rabbit nucleus pulposus (NP) and anulus fibrosus (AF) were cultured in alginate beads for as long as 14 days to allow them to reform a matrix made up of two compartments: the cell-associated matrix (CM) and further removed matrix (FRM). At different time points, the CM and FRM made by each cell population were analyzed using histologic, biochemical, and immunologic assays. OBJECTIVES: To study the metabolism of normal rabbit NP and AF cells in alginate by characterizing the CM and FRM formed by each cell population, and to identify metabolic properties that may shed light on mechanisms at play in disc degeneration. SUMMARY OF BACKGROUND DATA: Little is known about the metabolism of intervertebral disc cells, in part because of the lack of microculture systems appropriate for the study of these cells in vitro. In recent studies from our laboratories, it was suggested that articular chondrocytes cultured in alginate beads remain phenotypically stable and reform a matrix similar to the one they populate in vivo. This culture system appears ideally suited for the study of intervertebral cells available only in limited numbers. METHODS: Rabbit NP and AF cells released from the matrix by sequential enzyme digestion were encapsulated in alginate beads (20,000 cells/bead) and cultured for as long as 14 days. At selected time points, beads were solubilized with calcium chelating agents, and the CM and FRM were isolated. The rate of 35S-sulfate incorporation into proteoglycans, and the contents of various extracellular matrix molecules (total sulfated proteoglycans, antigenic keratan sulfate, hyaluronan, collagen, and pyridinium crosslinks) were measured. RESULTS: Both NP and AF cells remained phenotypically stable in the alginate gel throughout the culture period and reestablished a matrix composed of CM and FRM compartments. The two cell populations exhibited numerous differences in their metabolic activities in vitro. Nucleus pulposus cells synthesized fewer proteoglycan and collagen molecules and were less effective in incorporating these into the CM than AF cells. CONCLUSIONS: Intervertebral disc cells, especially NP cells, are extremely sluggish in reforming a CM, a protective shell rich in proteoglycans and collagen molecules. This may help explain why damage to the NP often is accompanied by progressive degeneration of the disc in vivo.
The extracellular matrix synthesized by articular chondrocytes cultured in alginate beads was investigated. Collagen levels increased sigmoidally with time and remained constant after 2 weeks of culture. The presence of cartilage-specific type II collagen was confirmed immunohistochemically. Predominantly type II collagen was present in the alginate bead, as reflected by the unique extent of lysyl hydroxylation, glycosylation, and pyridinoline crosslink formation measured. Collagen crosslinks, predominantly hydroxylysylpyridinoline (> 93%), were observed after 7 to 11 days of culture and their formation was effectively blocked by beta-aminopropionitrile (BAPN). Unexpectedly, BAPN treatment resulted in a 100% increase of collagen levels, without influencing cell proliferation and proteoglycan levels. In control cultures 90% of the synthesized collagen was retained in the cell-associated matrix, while in BAPN-treated cultures half of the collagen was found in the interterritorial matrix compartment further removed from the cells. This suggests that impaired crosslinking of collagen interferes with pericellular collagen deposition, causing upregulation of collagen synthesis by impaired cell-matrix interactions. Integrins are likely to be involved in this feedback inhibition by extracellular collagen since the cyclic RGD-containing peptide CGRGDSPC downregulated collagen synthesis by 37%.